The opioid crisis highlights the urgent need for novel non-addictive pain medications, as well as improved treatments for opioid addiction and overdose. There is a need for pharmacological agents directed at novel targets to test new therapeutic hypotheses, new clinic-ready drugs directed at those targets, and new testing systems with the potential to be more predictive of human clinical response than traditionally used models. This initiative proposes to develop and employ novel chemistry, screening, and testing methodologies to discover new pharmacological tools and investigational drugs for pain, addiction, and overdose. Remarkable advances in our understanding of the molecular and neural circuit physiology of pain, addiction, and overdose have suggested many putative approaches for therapeutic intervention. Very few of these are considered validated; most lack sufficient data to merit the considerable resources required to develop a new drug. Lack of validation is due largely to a combination of absence of drug-like pharmacological probes for novel targets which allow testing of new therapeutic hypotheses, lack of preclinical models sufficient to reliably predict human responses, and lack of market attractiveness (for treatments for addiction and overdose). This project intends to de-risk novel targets for pain, addiction, and overdose by creating preclinical pharmacological probes for novel targets. Over the past year, NCGC has created a comprehensive annotated HEAL library for strategic screening of compounds related to pain, addiction, and overdose. Specifically, the NCATS HEAL Library is aimed to 1) assemble all known and potential targets related to addiction or pain, 2) collect all known drugs, chemical probes, and active compounds in literature for the HEAL targets; 3) establish collaborations for drug repurposing, innovative profiling, hypotheses testing of tool compounds; 4) implement an integrated platform of HEAL project resource for data mining, machine learning, in silico modeling and virtual screening. Currently, the HEAL library consists of 3000 compounds associated with approximate 50 known and hypothesized HEAL targets, which have been collected from a diversity of resource, including public database (such as DrugBank, ChEMBL, IUPHAR, PubChem), NCATS in-house screening library (such as NPC, NPACT, MIPE), as well as target-focused HTS libraries from commercial vendors (such as MCE, Selleckchem). The library compounds and targets will be periodically updated and expanded. Of the 3000 compounds collected, physical sample of 2500 compounds have been acquired. For interesting compounds not available commercially, custom synthesis will be made by NCATS chemists or via outsourcing. All available compounds will be plated in qHTS 1536-well plate format, thus enabling a rapid and large-scale screening against a wide range of assays. The NCATS HEAL library is a public resource. A web-based platform will be implemented, providing access to the abundant information of the annotated HEAL targets and compounds, as well as the valuable assay screening data generated for various HEAL projects at NCATS. NCATS has multiyear experience performing quantitative high-throughput screening (qHTS) that enables generation of dose response curves for every compound, thus providing unique and rich datasets. Using cheminformatics tools and counterscreen/confirmatory assays, NCGC medicinal chemists are collaborating with partnering investigators to optimize promising compounds to improve potency, selectivity, and pharmacokinetic properties. These probe compounds are being further optimized through testing in advanced efficacy assays, with a view to moving these leads closer to the clinic as future therapies for pain, addiction, and overdose.